Much of the background information for this invention was described in British Patent Application GB 92 18482.9 by A. E. Dixon and S. Damaskinos, entitled "Apparatus and Method for Scanning Laser Imaging of Macroscopic Specimens". That application describes prior art systems for both fluorescence and photoluminescence imaging of large specimens using scanning stage confocal and non-confocal laser microscopes, and using camera systems in which the whole specimen is illuminated and imaged at the same time.
One type of large fluorescent specimen mentioned in GB 9218482.9 is the fluorescent gels used in DNA mapping and sequencing. A confocal scanning-stage laser fluorescence microscope was first described by Cox.sup.1, and the use of such a microscope to image fluorescent gels is described by Mathies and Peck in U.S. Pat. 5,091,652. Although this method has good spatial resolution, it is slow since the large fluorescent gels must be translated under the fixed laser beam, and the scan speed is limited by the speed of the moving stages. FNT 1. Cox, I. J. "Scanning optical fluorescence microscopy", Journal of Microscopy 133, 149-154 (1984).
In GB 9218482.9, Dixon and Damaskinos disclosed several embodiments of a scanning beam imaging system using a laser scan lens to focus the incoming laser beam onto the specimen, and then using the same lens to collect the reflected light (or photoluminescence or fluorescence) returning from the specimen. Most laser scan lenses are designed to give a constant scan velocity on the focal plane when the angle of deflection (theta) of the incident beam is varied at a constant rate. Such lenses are called F Theta lenses, and the image height is proportional to f*theta, whereas the image height of an ordinary photographic objective, or of a microscope objective, is f*tan(theta). Here theta is the angle between the incoming beam and the optic axis of the scan lens (the scan angle), f is the focal length of the laser scan lens, and * denotes multiplication. Some laser scan lenses are telecentric, i.e. they arc made in such a way that the cone of light converging toward focus at a spot in the focal plane is perpendicular to the focal plane for all scan angles.
One embodiment of the scanning beam imaging system disclosed in GB 9218482.9 is shown in FIG. 1. Light beam 103 from laser 102 (or other light source) is focused on pinhole 106 by lens 104. The expanding beam exiting pinhole 106 is focused to a parallel beam by lens 108. (Lens 104, pinhole 106 and lens 108 constitute a spatial filter and beam expander.) The parallel, expanded beam passes through beamsplitter 112 and is deflected in the x-y plane by first scanning mirror 114, which rotates about an axis parallel to the z-direction. The beam then passes through a unitary telescope comprised of lenses 116 and 118 and is brought back as a parallel beam to the center of second scanning mirror 120, which rotates about an axis parallel to the x-direction and imparts a deflection in the y-z plane. Lenses 122 and 124, also comprising a unitary telescope, return the deflected beam to the center of beamsplitter 126, which is placed at the position of the entrance pupil of laser scan lens 128. Light reflected from beamsplitter 126 is focused to a diffraction-limited spot on specimen 130 by laser scan lens 128. The scan system is controlled electronically to produce a raster scan of the focus spot across the specimen. Light reflected from (or photoluminescence or fluorescence emitted by) specimen 130 is collected by laser scan lens 128 and impinges on beamsplitter 126. Light passing through beamsplitter 126 is collected by condenser lens 132 and falls on the active area of non-confocal detector 134. Condenser lens 132 and non-confocal detector 134 comprise a non-confocal detection arm. Light reflected by beamsplitter 126 passes back through the scan system, and part of this returning beam is reflected by beamsplitter 112 towards detector lens 136 which focuses the returning parallel beam onto confocal pinhole 138. Light passing through the confocal pinhole is detected by confocal detector 140. Detector lens 136, confocal pinhole 138 and confocal detector 140 comprise a confocal detection arm. A confocal image of the specimen can be recorded pixel-by-pixel by digitizing the signal from confocal detector 140 using a slow-scan frame grabber synchronized to the mirror scan system. A non-confocal image can be recorded by digitizing the signal froth detector 134. In this embodiment of the scanning beam imaging system, laser scan lens 128 is a telecentric F Theta lens. The image from detector 140 is confocal, because Pinhole 138 rejects all light in the returning beam which is not parallel to the axis as it enters lens 136, and thus rejects all light that does not originate at the focal point of laser scan lens 128. The image detected by detector 134 is not confocal, and if beamsplitter 126 is a dichroic beamsplitter, then detector 134 can be used to detect fluorescence or photoluminescence from the specimen 130, without the reduction in intensity caused by the passage of the returning beam back through the scan system. This system works well in recording confocal images in reflected light, but performance is not as good in fluorescence and photoluminescence imaging, or for non-confocal imaging in reflected light. The amount of light emitted from the specimen in fluorescence or photoluminescence is usually small, and only a small part of that is collected by laser scan lens 128, since Laser Scan Lenses have a much smaller Numerical Aperture (NA) than most objective lenses used for fluorescence microscopes. In addition, most Laser Scan Lenses are not colour corrected, and they often provide diffraction-limited performance at only one wavelength, as well as having large changes in focal length with changes in wavelength. Thus, although detector 140 works well in reflected-light confocal imaging, the signal is very weak in confocal fluorescence or photoluminescence imaging, except from the brightest specimens. Colour-corrected Laser Scan Lenses are available, but they are complicated and very expensive. When using detector 134 for non-confocal reflected-light imaging, reflections from the lens surfaces inside the laser scan lens result in flare that degrades the image. In confocal reflected-light imaging, the confocal pinhole 138 is very, effective at reducing flare, as well as rejecting any light returning from the specimen that does not originate at the focal point of laser scan lens 128.